Recent evidence has showed that the tumor microenvironment (TME) may form a sanctuary for immune suppression and evasion. Although Immunotherapies based on checkpoint inhibitors and chemical modulation of the TME have garnered success, many challenges remain, including the heterogeneity of patient response and the serious side effects resulting from systemic autoimmunity. These observations suggested that biochemical stimuli are not the only factor that suppresses T cell functions within the TME. One emerging concept in the field is that the physical properties of the TME such as extracellular matrix stiffness, composition, and architecture also contributes to cancer cell proliferation and survival. However, whether the mechanical properties of the TME specifically modulates T cell activity, and thus contributing to immune evasion, remains unclear. There are two overarching goals for this proposal. First, to better understand the role of mechanical forces in T cell receptor activation, and T cell functional responses. Second, to understand how the physical aspects of TME affect T cell/tumor interactions and T cell function. My PhD work is focused on developing enabling technologies to study mechanobiology at the molecular scale, with a particular focus on the roles of mechanical forces in T cell activation. My work has shown that i) the T cell receptor transmits pN forces to its antigen during initial recognition, and in immunological synapse and ii) T cells use mechanical energy to discriminate antigens during the earliest step of T cell activation. For my remaining F99 phase, I will focus on investigating whether mechanical forces are important for T cell function. To achieve this goal, I will use recently developed proximity labeling technologies to identify mechanosensitive proteins that mediate T cell signaling. This strategy will allow for using proteomic analysis to determine the mechanical interactome (mechanome) in T cells. Then, I will determine whether mechanical forces affect long term T cell biological functions using ELISA and flow cytometry coupled with RNA-SEQ. Overall, the results from this integrative -omics study will offer better understanding of how T cells use mechanical energy to potentiate their biological functions. For my postdoc studies (K00 phase), I aim to understand how the physical aspects of TME affect cytotoxic T cell function (cancer killing). The goal is to quantify how ECM mechanics alter T cell function. This data will support the hypothesis that TME contributes to metastasis by enhancing immune evasion through physical mechanisms. The significance of this work pertains to developing new strategies for promoting T cell anti-tumor response. I propose to work in a lab that employs 3D matrices that mimic the TME and to use these scaffolds for co-culture of T cells and tumor grafts. The work will better define the physical parameters of the matrix (e.g. the extent of hydrogel stiffness, physical cues composition, and architecture) that affect T cell/tumor interaction and T cell function. Collectively, the work from both F99 and K00 phases will provide new fundamental understanding on the physical basis of T cell functions at molecular level and cellular levels. The outcome of this research may offer new design principles for targeted cancer immunotherapies.